This invention relates to a method for the production of proteins.
The presence of a variety of physiologically active proteins such as cytokines and peptide hormones has been ascertained and recent advances in genetic engineering technology are opening ways for large-scale production of these physiologically active proteins and clinical application of the same.
Interleukin-2 [hereinafter referred to as IL-2; also called T cell growth factor (TCGF)] is a lymphokine produced by T cells upon stimulation by a lectin or alloantigen, among others [Science, 193, 1007 1976)].
A large number of clones of killer T cells or helper T cells and, further, natural killer cells have so far been obtained through the utilization of IL-2 [e.g. Nature, 268, 154 (1977)]. In addition to such direct use in cloning T cells or natural killer cells, the use of IL-2 can result in selective in vitro proliferation of antigen-specific killer T cells capable of recognizing and destroying a certain particular antigen, for example a tumor antigen By introducing into animals tumor-specific killer T cells grown in this manner, it is possible to control or inhibit tumor growth [The Journal of Immunology, 125, 1904 (1980)].
These experimental findings suggest the possible utility of IL-2 as an antitumor agent It is further known that IL-2 restores the helper T cell function in nude mice which are deficient in thymus function [European Journal of Immunology, 10, 719 (1980)] and restores the induction of killer T cells against allogenic cells [Nature, 284, 278 (1980)], and therefore IL-2 can be expected to be useful in the treatment of immunocompromised diseases
Interferon-.alpha. (hereinafter referred to as IFN-.alpha.) and interferon-.gamma. (hereinafter referred to as IFN-.gamma.) are lymphokines produced by virus- or nucleic acid-activated lymphocytes, are biologically active in that they act on cells and bring them into an antiviral state, and thus play an important role in the prophylactic system or oncoimmune system.
Proteins such as these cytokines can be obtained as naturally occurring substances but in very limited amounts. However, recent advances in recombinant DNA technology have opened the way for the recovery of biologically active proteins from cultures of those strains of Escherichia coli and so forth which respectively carry expression vectors with genes for said proteins inserted therein [for IL-2: Nature, 302, 305 (1983) and Nucleic Acids Research, 11, 4307 (1983); for IFN-.alpha.: Journal of Interferon Research, 1, 381 (1981); for IFN-.gamma.: Nature, 295, 503 (1982)].
Since, whether it takes place in a eukaryote or in a prokaryote, protein biosynthesis starts with the messenger RNA codon AUG (which corresponds to methionine,) it is possible that the product protein may possibly be either a molecular species having a methionine residue at the N-terminal end or a molecular species having no such residue or a mixture of the two. In fact, it is known, for instance, that in Escherichia coli, the N-terminal end of many cell proteins is methionine [Conn & Stumpf: Outlines of Biochemistry, 4th edition, John Wiley & Sons (1976)] and that the initiation factor IF-3 of Escherichia coli comprises both the molecular species having a methionine residue at the N-terminal end and the species free of such residue [Hoppe-Seyler's Zeitschrift fur Physiologische Chemie, 354, 1415 (1973)]. With regards to proteins produced in Escherichia coli by using recombinant DNA techniques, it is known that the percentage of a addition of methionine residue to the N-terminal end is about 50% for IFN-.alpha. [Journal of Interferon Research, 1, 381 (1981)] and as high as 100% for human growth hormone [Nature, 293, 408 (1981)]. However, no instances have so far been reported to the control of the percentages of methionine residue addition percentage in such proteins
In the course of their investigations concerning the process for producing the IL-2 protein using strains of Escherichia coli with the IL-2 gene introduced therein, the present inventors found that the IL-2 protein produced in Escherichia coli is comprised of two molecular species, namely an N-terminal methionine residue-free IL-2, that is a molecular species beginning with an alanine residue as the N-terminal amino acid [Ala-IL-2], and a molecular species having a methionine residue added to the N terminal end and thus beginning with a methionyl-alanine residue [Met-Ala-IL-2], the content of the latter being much higher than that of the former.
Similarly, it was found that when IFN-.alpha. and IFN-.gamma. are produced in Escherichia coli each is a mixture of a molecular species the N-terminal end of which begins with a cysteine residue [Cys-IFN-.alpha. and Cys-IFN-.gamma., respectively] and a molecular species having a methionine residue added to the N terminal and thus beginning with a methionyl-cysteine residue [Met-Cys-IFN-.alpha. and Met-Cys-IFN-.gamma., respectively], the latter accounting for from 5-50%.
Those proteins which have a methionine residue at the N-terminal end are supposed to be similar in biological activity to the corresponding proteins of the naturally occurring type but, in any event, are different substances from the latter. Therefore, the known methods are not fully satisfactory for producing proteins having the respective amino acid sequences of the naturally occurring type protein.